In vitro evaluation of the effects of anti‐fungals, benzodiazepines and non‐steroidal anti‐inflammatory drugs on the glucuronidation of 19‐norandrosterone: implications on doping control analysis

Abstract

We have studied whether the phase II metabolism of 19-norandrosterone, the most representative metabolite of 19-nortestosterone (nandrolone), can be altered in the presence of other drugs that are not presently included on the Prohibited List of the World Anti-Doping Agency. In detail, we have evaluated the effect of non-prohibited drugs belonging to the classes of anti-fungals, benzodiazepines, and non-steroidal anti-inflammatory drugs on the glucuronidation of 19-norandrosterone. In vitro assays based on the use of either pooled human liver microsomes or specific recombinant isoforms of uridine diphosphoglucuronosyl-transferase were designed and performed to monitor the formation of 19-norandrosterone glucuronide from 19-norandrosterone. Determination of 19-norandrosterone (free and conjugated fraction) was performed by gas chromatography - mass spectrometry after sample pretreatment consisting of an enzymatic hydrolysis (performed only for the conjugated fraction), liquid/liquid extraction with tert-butylmethyl ether, and derivatization to form the trimethylsilyl derivative. In parallel, a method based on reversed-phase liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization with acquisition in selected reaction monitoring mode was also developed to identify the non-prohibited drugs considered in this study. Incubation experiments have preliminarily shown that the glucuronidation of 19-norandrosterone is principally carried out by UGT2B7 (39%) and UGT2B17 (31%). Inhibition studies have shown that the yield of the glucuronidation reaction is reduced in the presence of the anti-fungals itraconazole, ketoconazole, and miconazole, of the benzodiazepine triazolam and of the non-steroidal anti-inflammatory drugs diclofenac and ibuprofen, while no alteration was recorded in the presence of all other compounds considered in this study. Copyright © 2015 John Wiley & Sons, Ltd.

Drug-drug interactions and masking effects in sport doping: influence of miconazole administration on the urinary concentrations of endogenous anabolic steroids

“…In particular, miconazole is suspected to exert inhibitory activity on CYP450-dependent enzymes involved in steroid metabolic pathways (decrease in hydroxylated steroid formation) and on phase II metabolic conjugation with glucuronic acid. According to what in vitro demonstrated for other steroids [13,16], miconazole administration could be responsible for a decrease in Andro and Etio glucuronidation that reflects in the reduction of concentrations of these steroids in human urine.…”

“…Because it has been demonstrated that 19-NA can be endogenously produced [15], a threshold of 2 ng/mL (to be normalized for the specific gravity of urine) has been established. In this regard, the potential confounding/masking effect of DDIs, based on the in-vitro alteration of phase II metabolism of 19-nortestosterone by non-prohibited drugs (e.g., ketoconazole and miconazole), has also been recently reported [16].…”

“…T glucuronide (G), AndroG, EtioG] and of the T/E ratios [38,39]. This effect was ascribed to the inhibition of the 17a-hydroxylase and 17, 20-lyase activities of CYPC17 (conversion of pregnenolone to DHEA and progesterone to ASD) [40], but recent studies have also shown its inhibitory potential on steroid glucuronidation in vitro [13,16]. Figure 1 schematizes androgen biosynthetic and excretion metabolic pathways and underlines the sites of metabolic modulations by ketoconazole and finasteride [41].…”

“…In the previous studies we reported that, similarly to ketoconazole, the azolic antifungal miconazole is also able to inhibit UDP-glucuronosyltransferase and CYP enzymes during in vitro steroid metabolism [13,16], thus stressing the need to consider this kind of DDI in the evaluation of the analytical results in anti-doping analysis. Furthermore, other investigators reported the strong inhibitory effect of miconazole on CYP2C9 and 2C19 as well as on hepatic CYP3A4, 1A2, and 2D6 in vitro [42,43].…”

Effect of non‐prohibited drugs on the phase II metabolic profile of morphine. An in vitro investigation for doping control purposes

“…The in vitro phase II metabolism protocol was optimized starting from the procedures reported in literature . Different solvents (methanol, dimethylsulfoxide, and acetonitrile), buffers (phosphate and tris‐HCl), pHs of the incubation medium (5.0, 7.4, 8.0), concentrations of the substrate (0.01 to 10 mM) and of the enzymatic protein (0.1, 0.2, 0.5, and 1.0 mg/mL of either HLM or UGTs) and different incubation times (5, 10, 20, 30, 60, and 120 minutes) were tested.…”